1. Field of Use
The invention is directed towards the automated synthesis of polymers. More specifically, the invention relates to an apparatus for serial, continuous polymer synthesis, capable of rapid and low cost production of a large number of oligonucleotides.
2. Field of Use
Articles and publications set forth in this patent disclosure are presented for the information contained therein; none of the information is admitted to be statutory prior art and we reserve the right to establish prior inventorship with respect to any such information.
Oligonucleotides are used extensively in modem biotechnology. Applications for oligos include investigations relating to gene expression, polymorphism, drug discovery, and diagnostics, to name a few.
Arrays require large number of oligos, on the average of 10,000 per array. Oligo synthesis is vitally important for investigations using arrays. Oligos may be synthesized in situ, or "grown" where they are subsequently to be use, or oligos synthesized offline, deposited and attached to an array substrate. Such offline synthesis is commonly referred to as the synthesis of "whole" oligos. While automated DNA synthesizers exist, the time required to produce 10,000 oligos for an array is hundreds of hours. A batch type process and apparatus (see, for example, U.S. Pat. No. 5,814,700) can produce 8 dozen 25 mer oligo about every 2.5 hours. Shorter or longer lengths of oligos may be produced, but the batch process does not facilitate dramatic compression of synthesis times. Moreover, automatic synthesizers in use are not amenable to producing small amounts of oligos. Automatic synthesizers typically produce on the order of 200 Pico moles of an oligo, which is enough oligo for more than 400,000 arrays! Few applications require that many identical arrays. Often the actual amount required may be 40 nanomoles or less. The minimum quantities produced by automatic synthesizers result in much wasted oligos and all materials, included reagents and solvents, used in oligo synthesis.
Synthesizers such as the ABI, use columns as the support for the synthesis. Typical machines can support one to four columns at a time. Some others can have up to twenty-four in parallel. These systems are a closed flow-through operation All reagents are tied together to a common manifold. Both time and reagent are spent to flush our prior reagents switch to another reagent, and flow enough material through the column. Batch operation machines, such as described by Brennan (U.S. Pat. No. 5,814,700) use an array of wells, such as a 96 well titer plate. A solid support is inserted into each well and reagents are dropped into each well, and a differential pressure is applied from the top to the exit port at the bottom of the wells. The solution flows through the solid support to waste. Each line is dedicated to a single reagent, thus ensuring that the required amount will be delivered to the wells. A disadvantage is the time required dispensing reagent to each of the wells. Although reagent can be dispensed in parallel by replicating dispensers, the system then bears added complexities. Moreover, in apparatus in which reagents are dispensed serially, dwell times are different for the wells at the beginning of the titer plate versus wells at the end. Exposure times are not identical across rows. All synthesis chemistry is not equally tolerant of such differences, and the synthesis may be adversely affected.
Further, in batch mode, addition of each reagent must be complete before removal may be commenced.
As concerns scaling up the oligo synthesis process, merely adding more wells to a batch process, while increasing throughput, does not increase productivity. The more wells, the more time required to add reagents, or, alternatively, the more added reagent dispensing capability increases the complexity of the system.
At the most practical level, adding more reagent addition nozzles is hindered by close array spacing in denser arrays. Although approaches, such as adding a Y actuator, could be added to denser arrays, dispensing and actuation time become increasingly problematic.
What is needed is a scalable oligo synthesis apparatus capable of quick and cost effective oligo synthesis suitable for use in applications such as arrays.